Cultures of living organisms and tissue have recently become of extreme commercial and scientific value. Specialized bacteria have been developed for a wide variety of purposes, such as catabolism of organic waste products and preparation of materials for production. Other organic cultures, such as cell tissue cultures, have commercial value relating to testing of pharmaceuticals, vaccine and antibiotic production and in other usages.
Need for effective storage and preservation techniques has arisen concurrently with the increasing utilization of organic organism and tissue cultures. Various devices and methods have been utilized in attempts to maximize retention of tissue viability while minimizing cost, hazards and handling difficulties.
The most commonly used storage techniques relate to cryogenic methods. Organic tissue may be effectively reconstituted after quick freezing if proper procedures are followed. The best results have been obtained using liquid nitrogen (LN.sub.2) as the cryogenic medium. Nitrogen condenses from a gas to a liquid at -195.8.degree. C. (77.4.degree. K.,-320.4.degree. F.). Thus cultures stored in LN.sub.2 are subjected to extremely cold temperatures relative to ordinary conditions.
Various difficulties follow from the use of LN.sub.2 as a cryogenic medium. Initially, many common storage container materials are unsuitable for use at such low temperatures. Therefore it is necessary to select materials for culture containers which can withstand the extreme temperature ranges from room and incubation temperature to the liquid nitrogen storage conditions. In addition to fragility at low temperatures, the materials's thermal expansion characteristics may also cause problems. In most cryogenic and other viable tissue applications it is mandatory that a complete seal be maintained at all times. Leakage may cause contamination or extinction of the sample. Thus a container must be constructed in such a manner and of such materials that contraction and expansion of the materials do not lead to any leaks.
A further consideration in construction of cryogenic vials relates to strength under pressure gradients. Gases and liquids contained in the vials will significantly contract and expand in response to temperature changes. Since the volume of the vial remains relatively constant, the internal pressure will vary considerably. Thus the container walls and seal must be strong enough to withstand the pressure gradients.
Another consideration is the necessity that the vessels be sterilizable so that the cultures are not contaminated. This is especially important if the containers are intended to be used more than once.
The traditional containment vessels for cryogenic preservation of cultures have been glass vials which are filled and then sealed by melting the aperture shut. The glass vials are not entirely satisfactory since they are not reusable. Furthermore, inherent weaknesses in the vessel walls in the vicinity of the seal can often lead to leaks and or explosions. It is not unusual for 10% of a collection of samples to be lost upon thawing due either to explosions or to vessel leakage which destroys the viability of the sample. Nonetheless, the failure of alternate vessels to solve the other problems inherent in cryogenic storage has resulted in the continued usage of disposable glass vials.
Various attempts have been made in the art to develop alternate storage vessels for cryogenic uses. Plastic vials having exterior threads for receiving a cap are manufactured by the Wheaton Company. Vials having interior threads are distributed by A/S Nunc of Denmark and Dynatech. Each of these vials may be sealed. These vials use the threads as the primary seal, although the Nunc devices also include a gasket as a secondary seal. The prior art vials are less than satisfactory in one or more ways in that they are subject to cracking, breakage and leakage and may also be complex and expensive to manufacture.
The present inventors are unaware of any existing patents dealing specifically with cryogenic containers or methods for sealing. However, containers and seals adapted for similar purposes have been the subject of several prior patents.
U.S. Pat. No. 3,032,225, issued to S. Harding disclosed a closure for a sealed vessel including a disposable inner seal and an outer screw cap. U.S. Pat. No. 3,860,135 issued to a Yung, et al, discloses a container with an attached cap. Dual element sealing caps are also disclosed by U.S. Pat. Nos. 3,804,284 issued to Moore, et al and 3,877,598, issued to Hazard. Additional sealing means are disclosed in U.S. Pat. Nos. 4,211,33, issued to Villarejos and 2,987,175, issued to E. W. Bottum.
The Harding disclosure is particularly adapted for use on soft drink bottles and similar containers. It envisions a disposable metal inner cap which is molded about the top of the container. An outer threaded cap is then placed on the bottle over the inner cap. A central depression in the caps causes the inner cap to be forced into the bottleneck and thus, increases the integrity of the seal. The inner cap is destroyed upon opening and cannot be reused. The Harding sealing method would not be applicable to cryogenic storage since the use of different materials for the inner cap and the bottle, required for the Harding technique to work, would result in unavoidable gaps in the seal caused by nonuniform thermal expansion characteristics.
The Moore et al device also utilizes a separate inner cap. The Moore et al device is adapted for large, complex applications and the inner cap is intended to be substantially deformed during use. The device is not appropriate for cryogenic use due to its material requirements and complexity.
None of the prior art methods solve the various problems associated with cryogenic storage of living organic organisms and tissue under a tight seal in economical, simple and reliable manners.